N-acetylcysteine prevents 4-hydroxynonenal- and amyloid-β-induced modification and inactivation of neprilysin in SH-SY5Y cells

As one of the dominant amyloid-β peptide (Aβ) proteases, neprilysin (NEP) plays a crucial role in maintaining a physiologic balance between Aβ production and catabolism. We have previously shown that NEP is modified by 4-hydroxynonenal (HNE) adducts, resulting in decreased activity in the brain of AD patients and cultured cells. In order to determine whether antioxidants can rescue NEP, SH-SY5Y cells were treated with HNE or Aβ, together with N-acetylcysteine for 24 hours, prior to analysis of NEP protein levels, activity, and oxidative modifications. Intracellular NEP developed HNE adducts after 24 hours of HNE or Aβ treatment as determined by immunoprecipitation, immunoblotting, and double immunofluorescence staining. N-acetylcysteine at 10 to 100 μM alleviated HNE adduction after HNE or Aβ treatment. In keeping with previous reports, HNE-modified NEP showed decreased catalytic activity. The present study demonstrates that antioxidants can be used to spare NEP from oxidative modification, suggesting a potential mechanism underlying the neuroprotective effects of antioxidants in aging or Alzheimer's disease.